B-1 B cells are part ofthe innate immune system and arise from distinct B-1 progenitors that preferentially emerge in the fetus. The focus of the current application has been to characterize B-1 progenitors and contrast their development to that of B-2 B cells. During the past 3.5 years, we have identified the fetal yolk sac and para-aortic splanchnopleura as sites in which B-1 progenitors arise prior to the emergence of B-2 progenitors, demonstrated that the B-1 and B-2 lineages diverge by the common lymphoid progenitor stage of development, showed that the transcriptional regulation of B-1 and B-2 progenitor development is distinct, and identified B-1 transitional cells in the spleen that, in contrast to B-2 transitional cells, do not require engagement of the BAFF-receptor or activation of the alternative NF-KB pathway in order to survive or mature. These data have allowed us to formulate a model in which the first waves of B cell development that occur in the fetus preferentially generate B-1 progenitors. Our aims for the next funding period will continue to interrogate this model and build upon it. Aim 1 will develop an in vitro, murine embryonic stem cell differentiation system in which the fetal B-1 waves are recapitulated, and this will be used to identify the cellular intermediates from which B-1 progenitors derive. Aim 2 will compare patterns of gene expression in B-1 and B-2 progenitors in order to identify genetic signatures that distinguish these two lineages and use in vivo and in vitro models to assess the role of specific genes in B-1 progenitor emergence and maturation. Aim 3 will extend these studies to the human in order to identify human fetal B-1 progenitors. Because B-1 progenitor production is most robust in the fetus and neonate, which is also the time when exposure to antigens regulates the formation ofthe mucosal immune system; we hypothesize that the gut is seeded by B-1 cells generated during the fetal/neonatal window of development and will test this in Aim 4. Taken together, these data will provide new insights into the development of the humoral immune system in the fetus and neonate of both mice and humans. RELEVANCE (See instructions): B-1 cells provide protection against specific pathogens, such as encapsulated bacteria, and they are also though to be involved in some autoimmune diseases. We present data in our progress report suggesting that transformed B-1 progenitors may also initiate aggressive forms of B-acute lymphoblastic leukemia. Thus, studies that further our understanding of B-1 development have direct relevance to human health.